The research outlined in this proposal deals with the broad topics of synthesis and transport of lipid molecules within mammalian cells, and is important for a proper understanding of membrane biogenesis. The following areas will be investigated. We will continue our studies with fluorescent lipid analogues which permit traditional lipid biochemical analyses to be performed and correlated with information about the intracellular location of lipid metabolites determined by fluorescence microscopy in living cells. The general applicability of this approach will be examined in parallel studies using isotopically labeled lipids and EM autoradiography. We will study the internalization and degradation of both radiolabeled lipids and fluorescent lipid analogues after insertion into the plasma membrane of cells by lipid transfer from liposomes. These data will be compared to the rate of appearance of newly synthesized lipids at the cell surface to determine if recycling of plasma membrane lipids occurs. We will study the behavior of fluorescent lipids, liposomes, and other agents after microinjection into single cells. These experiments are designed to develop an in situ approach for studying the compartmentalization of lipids during their biosynthesis and for probing mechanisms of lipid transport within individual cells. Finally, using a selection procedure based on fluorescence photobleaching, we will attemtp to isolate mutant cells which are defective in their ability to incorporate, metabolize, and/or translocate the fluorescent lipid analogues among intracellular membranes. The studies listed above are basic to an understanding of membrane assembly in eukaryotes, and are fundamental to the development of rational treatments of membrane or cell surface-related disease states.